Mechanical transfection devices and methods

ABSTRACT

Systems and methods for transfection devices are contemplated for delivery of various complex macrostructures. Preferred systems and methods are suitable for mRNA reprogramming and genome editing and use mechanical force to induce uptake of the macrostructures in a target cell. Contemplated devices are able to achieve high throughput of transfected cells in remarkably short time that remain viable and are capable of producing colonies.

This application is a continuation-in-part application of co-pendingU.S. application with the Ser. No. 14/754,625, filed Jun. 29, 2015,which claims priority to the U.S. provisional application with Ser. No.62/020,910, filed Jul. 3, 2014. This application further claims priorityto U.S. provisional application with Ser. No. 62/049,747, filed Sep. 12,2014.

FIELD OF THE INVENTION

The field of the invention is cellular cargo delivery technologies andmethods therefor.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications identified herein are incorporated by reference to thesame extent as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

Transfection processes are used to deliver various types of materialsinto a cell, and there are numerous known methods in the art. Forexample, U.S. Pat. No. 5,586,982 describes a treatment device capable ofdelivering genetic material or drugs into cells of a patient in vivousing heat to assist with transfection. Unfortunately, such approachoften tends to damage the cells. Moreover, since the poration lasts onlyfor a very short time, the amount of material delivered into the cellswill in most cases be significantly reduced, especially where thematerial is relatively large. Finally, such approach also fails toprovide a method for culturing cells after transfection.

In another example, as described in US 2009/0081750, magnetic fields areemployed to move cells through a channel in which the cells undergotransfection. Actual transfection is then performed via several possiblemanners, including electroporation, heat, or light. Similar to the '982reference, effective transfection is typically limited to relativelysmall molecules and low quantities. Yet another example of poration totransfect cells is described in WO 2013/059343. Here, cells are fedthrough a microfluidic channel in a buffer that contains a deliverymaterial. The cells pass through a constriction region, which causes thecells to become perturbed with pores through which the delivery materialthen diffuses. While this approach overcomes to at least some degreeissues associated with short pulse time, delivery still requiresporation.

A more extreme approach is presented in U.S. Pat. No. 5,858,663 in whicha cold gas shockwave is used to accelerate micro projectiles that carrymatter into the cells. While such approach guarantees delivery of evenrelatively large molecules into a cell, it is readily apparent that suchapproach is also prone to significantly damage a cell.

WO 96/24360 attempts to overcome shockwave damage by providing atime-dependent impulse transient characterized by rise time andmagnitude that is thought to increase the overall permeability of a cellmembrane, which results in an increase in diffusion of materials intothe cell. The impulse is achieved by applying an optical field to a filmon which the cells are grown, and the optical field ablates the filmthereby delivering the impulse. While such approach will provide fortransfection, high throughput production of transfected cells remainsproblematic. To increase throughput, WO 02/42447 teaches use ofleverages shock or other forms of pressure, and U.S. Pat. No. 7,687,267describes a high throughput cell transfection device for transfer ofsmall nucleic acid molecules (e.g., DNA, siRNA) through electroporationwhere the device contains an array of cell transfection units.Similarly, US 2012/0244593 teaches a high throughput electroporationtransfection device, which requires poration (i.e., electroporation) anddiffusion to deliver the material.

Unfortunately, the known transfection devices require significantdisruption to a cellular membrane to allow for greater diffusion ofcargo material, which becomes especially difficult where the cargomaterial is relatively large. For example, clustered, regularlyinterspaced, short palindromic repeat (CRISPR) technology has emerged asan important tool for performing targeted and highly-efficient editingof a cell's endogenous genome as evidenced by Cong, L. et al. “MultiplexGenome Engineering Using CRISPR/Cas Systems”, Science 339, 819-823(2013); and Ran, F. A. et al. “Genome engineering using the CRISPR-Cas9system”, Nature Protocols 8, 2281-2308 (2013). The two essentialcomponents of CRISPR technology are a guide RNA and a RNA-guidednuclease, e.g. Cas9. The guide RNA specifies the targeted DNA sequencewhile recruiting the Cas9 nuclease to the target site for gene editing.Advantageously, multiple genes can be targeted simultaneously bydelivering a multiplex of different guide RNA sequences.

One especially desirable use of such complex gene editing is thegeneration of induced pluripotent stem (iPS) by co-expressing of acocktail of transcription factors in differentiated somatic cells to soreprogram the cells into a pluripotent state (see e.g., Takahashi, K. &Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonicand adult fibroblast cultures by defined factors”, Cell 126, 663-676(2006)). Using mRNAs encoding the respective reprogramming factors, iPScells can be generated without leaving a genetic footprint in thereprogrammed cells with higher efficiency compared to other DNA-basedapproaches as indicated by Warren, L. et al. “Highly EfficientReprogramming to Pluripotency and Directed Differentiation of HumanCells with Synthetic Modified mRNA”, Cell Stem Cell 7, 618-630 (2010);and Mandal, P. K. & Rossi, D. J. “Reprogramming human fibroblasts topluripotency using modified mRNA”, Nature Protocols 8, 568-582 (2013).

However, transfection efficiency and viability using multiple distinctnucleic acids is often problematic and requires in all or almost allinstances multiple steps during which cells need to be transferred orotherwise manipulated. As a consequence, currently known transfectionmethods often require significant time to generate iPS cells, andviability and/or yield is often much less than desired.

Therefore, there is a need for improved transfection devices and methodsfor delivery of cargo of various sizes, and especially large and/ormulti-component cargo into a cell in a manner that will not or onlyminimally adversely affect the cell.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to systems and methods fortransfection devices in which a macrostructure, a complexmacrostructure, or multiple different macrostructures are deliveredunder pressure from a deformable fluid reservoir to cells contained in acontainer. Most preferably, the deformable fluid reservoir is coupled tothe container via a microporous membrane having a pore size suitable todeliver the macrostructure to the cell, which is thought to internalizethe macrostructure via active endocytosis. Such devices arecharacterized by their high transfection efficiency, even with complexor multiple macrostructures, and low rate of cell damage.

In one aspect of the inventive subject matter, a method of changinggenetic content of a cell that includes a step of providing a pluralityof cells and exposing the cells to a transfection medium comprising atransfection reagent in association with a plurality of distinct nucleicacids. Most typically, the step of exposing the cells includes a step ofapplying pressure to the transfection medium to thereby force thetransfection medium against the cells for a time and at a pressureeffective to introduce the plurality of distinct nucleic acids into thecells. In a subsequent step, the cells are cultivated for a timesufficient to integrate or express the plurality of distinct nucleicacids. Additionally, it is contemplated that the steps of exposing andcultivating are performed in the same container containing the pluralityof cells, typically for a time sufficient to allow for generation ofclonal daughter cells. Therefore, extra manipulation steps on the cellsare typically not required.

While not limiting to the inventive subject matter, it is generallypreferred that the step of exposing the cells is non-ballistic and thatthe pressure to the transfection medium is at least 100 hPa. Typically,the pressure to the transfection medium is maintained for a period ofbetween 10 ms and 30 s, and the cells are retained in a fixed position(e.g., microfluidic channel or a cell adhesive layer) during the step ofexposing and/or application of pressure.

In some contemplated aspects, the cells are typically located on aporous membrane during the step of exposing, and the transfection mediumis forced through pores of the porous membrane. For example, the porousmembrane may have an average pore density of between 1×10⁶ pores/cm² and1×10⁸ pores/cm², and/or an average pore size of between 0.5 μm and 10μm.

In other contemplated aspects, the change in genetic content reprogramsa differentiation stage of a cell (e.g., from fully differentiated cellto induced pluripotent stem cell) or edits genomic information of a cell(e.g., via homology directed repair). With respect to the transfectionmedium it is contemplated that the medium comprises cationic liposomes,and/or that the plurality of nucleic acids comprise a guide RNA, aregulatory RNA (e.g., siRNA, miRNA, antisense RNA, etc.), and/orplurality of distinct RNAs that encode respective different proteins.Thus, the RNAs encode genes suitable for reprogramming a differentiatedcell into a induced pluripotent stem cell (e.g., human cell).Alternatively, the plurality of nucleic acids may also comprise at leastone DNA and at least one RNA, most typically suitable for genome editing(e.g., Crispr/Cas).

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a schematic overview of one exemplary transfection deviceaccording to the inventive subject matter.

FIG. 1B provides a detail view of an exemplary stage and container ofthe device of FIG. 1A.

FIG. 1C provides a detail view of an exemplary deformable fluidreservoir (upper panel) and container engaged with the reservoir (lowerpanel) of the device of FIG. 1A.

FIG. 2 illustrates another exemplary configuration of an assembly of thecontainer and deformable fluid reservoir according to the inventivesubject matter.

FIG. 3 illustrates an exemplary detail in which a pneumatic actuator(left panel) or a mechanical actuator (right panel) interacts with thedeformable fluid reservoir.

FIG. 4 is a graph illustrating exemplary deflection of a wall in thedeformable fluid reservoir as a function of type and force applied tothe wall.

FIG. 5 is a graph depicting colony numbers as a function of pumppressure.

FIG. 6 is a graph depicting transfection frequency as a function oftransfection method.

FIG. 7 is a graph depicting colony forming frequency after delivery ofmitochondria as function of types of substrate and pore size.

FIG. 8 is a graph depicting colony forming frequency after delivery ofmitochondria as function of pore density.

FIG. 9 is a graph depicting pore density-normalized colony numbers as afunction of membrane materials and pore size.

FIG. 10 is a graph depicting transfection rates for DNA (upper panel)and RNA (lower panel) using nucleic acid lipoplexes indicating successof transfection of DNA (upper panel) and RNA lipoplexes (lower panel)using contemplated methods relative to known top loading techniques.

FIGS. 11A and 11B are graphs depicting colony numbers using contemplateddevices and methods as a function of mitochondria material (upper panel)and mitochondria pretreatment (lower panel).

FIGS. 12A and 12B are graphs depicting transfection rates for deliveryconditions (upper panel) and cell pretreatment (lower panel) usingcontemplated methods and devices.

FIGS. 13A and 13B are graphs depicting transfection rates (upper panel)and GFP intensities (lower panel) as a function of transfection agentsusing contemplated methods and devices.

FIG. 14 illustrates results for control and comparative transfections(Top vs. PASTe) of BJ fibroblasts with RNA encoding GFP.

FIG. 15 illustrates results for control and comparative transfections(Top vs. PASTe) of H9 embryonic stem cells with RNA encoding GFP.

FIG. 16 illustrates time course results for comparative transfections(Top vs. PASTe) of BJ fibroblasts with RNA to reprogram the cells toform iPS.

FIG. 17 is a schematic graph illustrating an exemplary approach forCRISPR gene editing using PASTe.

FIG. 18 is a graph depicting exemplary results for comparativetransfections (Top vs. PASTe) of HEK 293 cells.

FIG. 19 is a photograph showing effects of gene editing efficiency byhomology-directed repair (HDR) using EMX1.

DETAILED DESCRIPTION

The inventors have discovered methods and devices to deliver variousmacrostructures, including cellular components (e.g., various anddistinct nucleic acids, typically DNA and/or RNA in association with atransfection agent), cell organelles, and bacteria into a target cell ina non-disruptive manner. Moreover, contemplated methods and devicesallow a large number of target cells to be modified, thus allowing forhigh-throughput transfection.

Most notably, the inventors have discovered that a relatively moderatemechanical force applied to macrostructures and target cells will leadto uptake of the macrostructures into the cells in a non-destructivemanner, most likely via active endocytosis. Among other benefits, theinventors discovered that contemplated systems, methods, and devices areespecially suitable for delivery of complex and diverse cargo, andparticularly for gene editing and reprogramming of cells where multipleand relatively large nucleic acid molecules in complex or otherassociation with transfection agents such as cationic liposomes aretranslocated into the cells.

Therefore, in one aspect of the inventive subject matter a transfectiondevice for delivery of various macrostructures (e.g., mitochondria,bacteria, DNA/RNA lipoplexes, etc.) into target cells is contemplated inwhich an actuator provides a force that acts upon the macrostructures(typically in solution) and the cells such that the macrostructures thatare proximal to the cells are taken up into the cells. In especiallypreferred transfection devices, movement of the cells is at leasttemporarily restrained (or adherent cells are employed), and the cellsare initially separated from the macrostructures by a porous membrane,wherein the macrostructures are contained in a deformable fluidreservoir. Upon exertion of a force onto the deformable fluid reservoir,the macrostructures pass across the pores to the at least temporarilyimmobilized cells, triggering uptake of at least some of themacrostructures into the cells. It should thus be appreciated that thedelivery of the macrostructures (e.g., transfection reagent inassociation with various and distinct nucleic acids) will not be aballistic transfer in which a nucleic acid-loaded particle or droplet isshot at a sufficiently high velocity at a cell to ballisticallypenetrate the cell membrane. Instead, it is believed that the uptake isan active uptake process that is initiated or mediated by elevatedpressure and/or enhanced local contact (e.g., active endocytosis).

For example, contemplated transfection devices will typically have astage configured to receive and retain a container to which a deformablefluid reservoir is fluidly coupled. The stage is further configured toallow positioning of the container relative to the fluid reservoir suchas to allow movement of macrostructures contained in a fluid from thedeformable fluid reservoir into the container. In most preferredaspects, movement is facilitated via a porous membrane. The transfectiondevice will also include an actuator that is operably coupled to thestage and that is configured to allow exertion of a force onto at leasta portion of the deformable fluid reservoir in an amount effective tocause the movement of the macrostructure from the deformable fluidreservoir into the container. As used herein, and unless the contextdictates otherwise, the term “coupled to” is intended to include bothdirect coupling (in which two elements that are coupled to each othercontact each other) and indirect coupling (in which at least oneadditional element is located between the two elements). Therefore, theterms “coupled to” and “coupled with” are used synonymously.

One exemplary device is schematically illustrated in the detail view ofFIG. 1A. Here, the device 100 has a stage 110 that is configured toremovably retain one or more containers 112 that contain cells fortransfection. In the example of FIG. 1A, the bottom surface of container112 includes a porous membrane 114 and the lower portion of thecontainer 112 is configured such as to sealingly engage with the amating portion of the deformable fluid reservoir 120 as is shown in moredetail below. Base plate 130 is disposed below the deformable fluidreservoir 120 and includes one or more pistons 132. Actuator 140 isdisposed below base plate 130 and exerts an upwards force onto baseplate 130 such that the pistons engage with a lower surface of thedeformable fluid reservoir 120 as is also shown in more detail below.FIG. 1B provides a more detailed view of an exemplary stage 110. Here,the stage includes a receiving plate 113 having one or more openings toremovably receive one or more containers 112 that have a porous membrane114 at their lower surface. Container 112 is retained in the openings ofthe receiving plate via cover plate 111 such that when the actuator 140exerts force upon the deformable fluid reservoir 120, the containerremains in the same position relative to the stage.

The upper panel of FIG. 1C shows a detail view of an exemplarydeformable fluid reservoir 120 where the deformable fluid reservoircomprises multiple layers. Here, the top layer 121 comprises a pliablematerial that allows for sealing and retaining engagement with thecontainer (see lower panel). While not limiting to the inventive subjectmatter, the top layer will have cutouts and have a thickness that isselected such as to provide sufficient volume for a fluid that containsthe macrostructures when the container is sealingly engaged with thedeformable fluid reservoir. Thus, it should be appreciated that thefluid that contains the macrostructures is contained by both, thedeformable fluid reservoir and the porous membrane of the container.Middle layer 122 is typically made from a deformable material thattogether with the cutouts in the top layer forms a well for the liquidcontaining the macrostructures. In especially preferred aspects, thematerial for the middle layer is selected such as to allow a compressiveforce to act on the middle layer to thereby produce a fluid pressure ofat least 50 hPa, or at least 100 hPa, or at least 200 hPa, or at least400 hPa when the container is sealingly engaged with the container. Thebottom layer 123 of the deformable fluid reservoir typically provides arigid support platform for the middle and top layers and will typicallyinclude one or more openings for the actuator or piston 132 of the baseplate 130.

The lower panel of FIG. 1C schematically illustrates a container 112sealingly engaged (typically via press fit) with the top layer 121 suchthat the porous membrane 114 at the bottom of the container 112 is influid contact with the suspension 170 formed from the fluid and themacrostructures. Direct or indirect actuation (e.g., via piston 132)exerts mechanical force 141 onto the deformable middle layer 122. Middlelayer and top layer are both supported by bottom layer 132. Thus, itshould be appreciated that by providing a force onto the pliable middlelayer, pressure in the fluid space defined by the middle layer, the toplayer, and the porous membrane increases, leading to migration of themacrostructures through the pores across the porous membrane into thecontainer and cells. In the example of FIG. 1C, the cells are retainedat the bottom of the container (i.e., on the top side of the porousmembrane, not shown) by a thin layer of CELL-TAK™ adhesive (polyphenolicproteins extracted from the marine mussel, Mytilus edulis, commerciallyavailable from Corning Inc., Bedford, Mass.).

Another exemplary device is schematically shown in the detail view ofFIG. 2 where the container 212 is configured as a well of a multi wellplate. The deformable fluid reservoir in this device comprises a frameportion 225 that helps retain porous membrane 214. Also coupled to theframe portion 225 is a deformable bottom 226 of the reservoir. Thus, asnoted previously, the porous membrane and a deformable portion of thefluid reservoir define the fluid reservoir that contains themacrostructure, and pressure onto the deformable portion will result inpassage of the macrostructures through the pores of the porous membraneto the cells. Most typically, the fluid reservoir is preloaded, but itshould be noted that the fluid reservoir may also include one or morefill and vent ports to fill the fluid reservoir before use.

With respect to suitable containers it is generally contemplated thatthe container may be made from a variety of materials, however,especially preferred materials are sterilizable by heat, radiation,and/or chemical treatment. Therefore, appropriate materials includevarious polymers (e.g., PE, PET, HDPE, PDMS, PC, etc.), glass, metals,and all reasonable combinations hereof. Regardless of the material, itis further preferred that the container has a shape suitable forreceiving and retaining mammalian cells and that the container isconfigured to allow culturing of the cells. Thus, containerscontemplated herein will typically have a volume between 0.1 mL and 250mL, or even higher. For example, where the container is configured as amulti well plate, suitable volumes will be between 0.1 and 20 mL. On theother hand, where the container is configured as a culture flask orculture beaker, suitable volumes will be between 10 and 250 mL, orbetween 250 and 1000 mL. Thus, the shape of suitable containers istypically not limited and shaped considered suitable for use inconjunction with the teachings presented herein include cup shapes, cellculture flask shapes, box shapes, cylinder shapes, Petri dish shapes,etc. In still further contemplated aspects, containers will preferablybe single use and disposable containers that are sterilized.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Regardless of the particular shape and volume, it is contemplated thatthe container will comprise (or be fluidly coupled to) at least oneportion of a surface that includes the porous membrane. In most cases,the porous membrane forms at least a portion of a bottom surface of thecontainer. There are numerous porous membrane known in the art, and allporous membranes are deemed suitable so long as such porous membranesare able to support and/or retain cells during transfection. Forexample, contemplated porous membranes may be made from variousmaterials, including nylon, polytetrafluoroethylene (PTFE), expandedpolytetrafluoroethylene (ePTFE), polyetheretherketone (PEEK), expandedpolyetheretherketone (ePEEK), polyethylene (PE), polypropylene (PP),polyvinylidene fluoride (PVDF), ethyl vinyl acetate (EVA), thermoplasticpolyurethane (TPU), or polyethersulfone (PES). In further contemplatedaspects, the membrane will typically be relatively thin and maytherefore have a thickness of between 1 μm and 1 mm, or between 3 μm and0.5 mm, or between 5 μm and 250 μm. Viewed form a different perspective,suitable membranes will generally have a thickness of at least 1 μm, orat least 3 μm, or at least 5 μm, or at least 10 μm. It should be notedthat the membrane thickness will also be determined at least in part bythe pressure applied to the deformable fluid reservoir. However, supportstructures (e.g., support grid or mesh) to avoid membrane failure arealso expressly contemplated herein.

The average pore size of the porous membrane will typically depend onvarious factors and the size and/or flexibility of the macrostructurewill be particularly relevant. Therefore, it is contemplated that theaverage or median pore size of the porous membrane ranges from about 50nm, or from about 100 nm, or from about 200 nm, or from about 300 nm, orfrom about 400 nm, or from about 500 nm, or from about 600 nm, or fromabout 700 nm, or from about 800 nm, or from about 900 nm, or from about1 μm up to about 30 μm, or up to about 20 μm, or up to about 15 μm, orup to about 10 μm, or up to about 8 μm, or up to about 5 μm. In certainembodiments the median or average pore size in the porous membrane isabout 1 μm or about 3 μm, or about 5 μm or about 10 μm, or about 15 μm.The term ‘about’, when used in conjunction with a numeral refers to arange spanning +/−10%, inclusive, around that numeral. For example, theterm ‘about 10 μm refers to a range of 9 to 11 μm, inclusive.

With respect to the pore density of the membrane it is contemplated thatthe density will be sufficiently high such that on average a cell willcover (or be located above) at least one pore, or at least two pores, orat least 3 pores, or at least 4 pores, or at least 5 pores, or at least10 pores. Thus, the pore density in some embodiments will be betweenabout 1×10⁵ pores/cm² to about 1×10⁷ pores/cm², or between about 5×10⁵pores/cm² to about 5×10⁶ pores/cm², or at least 1×10⁵ pores/cm², or atleast 1×10⁶ pores/cm², or at least 1×10⁷ pores/cm². Viewed from adifferent perspective, the porous membrane comprises in some embodimentsabout a 1-10 μm diameter average pore size at about 1×10⁶-10⁷ pores/cm².

In still further contemplated aspects of the inventive subject matter,additional elements may be included with the container and membrane toat least temporarily retain cells in a fixed position on the porousmembrane. For example, additional elements may include microfluidicchannels though which the cells may be fed to/maintained on themembrane, a mesh to retain the cells on the membrane, or the membranemay be coated with an adhesive that temporarily retains the cells on themembrane. There are various adhesives known in the art, and all of themare deemed suitable for use herein, including collagen matrices,CELL-TAK™ adhesive, poly-L-lysine, extracellular matrix proteins (e.g.,collagen, fibronectin, laminin, etc.), or other adherents.Alternatively, it is noted that where adherent cells are used, noadditional elements to retain the cells may be needed.

Deformable fluid reservoirs contemplated herein will generally have avolume suitable for retaining sufficient macrostructures to transfect adesirable number of cells. Consequently, depending on the cell number,the transfection efficiency, surface area of the porous membrane, andother factors, the volume of the deformable fluid reservoir may varyconsiderably. However, it is generally contemplated that the volume willbe between about 10 μL and 5 mL (in some cases even higher), or betweenabout 100 μL and 1 mL, or between about 50 μL and 500 μL, or betweenabout 100 μL and 1000 μL. Similarly, the shape of the deformable fluidreservoir may vary considerably but it is generally contemplated thatthe particular shape will be at least in part determined by the shape ofthe container and the size of the porous membrane. Consequently, it iscontemplated that the deformable portion will preferably a wall or wallportion, which may be homogenously deformed or locally, or that thedeformable portion is replaced by a movable wall or wall portion (e.g.,configured as a plunger). Alternatively, the entire deformable fluidreservoir may also be compressible. Moreover, it is contemplated thatthe deformable fluid reservoir will sealingly engage with the containersuch that the macrostructures will be able to flow from the reservoir(typically within the fluid) into the container and to the cells on theporous membrane. In especially preferred aspects, sealing engagement ofthe deformable fluid reservoir with the container is maintained atpressures in the deformable fluid reservoir of at least 50 hPa, at least100 hPa, at least 200 hPa, at least 400 hPa, at least 800 hPa. Viewedfrom a different perspective, the sealing engagement may be maintainedat pressures between 1 and 1000 hPa, between 10 and 800 hPa, between 50and 600 hPa, or between 100 and 1000 hPa.

In still further contemplated aspects of the inventive subject matter,the deformable fluid reservoir may be coupled to or include or one ormore ports through which a fluid containing the macrostructures can beintroduced, preferably using sterile techniques and sterile adapters(e.g., Luer lock adapters). Where desirable, vent and/or discharge portsmay be included to facilitate loading and unloading of the deformablefluid reservoir. Most typically, the deformable fluid reservoir will beremovable. However, permanently affixed deformable fluid reservoirs arealso contemplated (in such case, the reservoirs may be prefilled with afluid and macrostructures).

Among other things, macrostructures suitable for use herein will includecell organelles (e.g., nucleus, mitochondria, chloroplast, ribosomes,etc.), viruses and microorganisms (e.g., gram⁺ and gram⁻ bacteria,etc.), various macromolecules, and especially recombinant and naturalnucleic acids alone or in combination with a transfection agent (e.g.,native, synthetic or artificial chromosomes, miRNA, siRNA, plasmids,double minute chromosomes, etc.), proteins and/or protein complexes, anddrug delivery particles.

Therefore, the type of fluids appropriate for storage and movement mayvary considerably, but it is generally preferred that the fluids includephysiologically acceptable solutions (e.g., isotonic and bufferedsolutions), growth media, etc. Examples of suitable fluids includeexperimental buffer, PBS, DMEM, HBSS, Opti-MEM, DMEM without Ca2+, orother media amenable to the nature of cargo material. Themacrostructures in the fluid can also comprise one or more lipidcarriers. Example lipid carries can Lipofectamine, Transfectace,Transfectam, Cytofectin, DMRIE, DLRIE, GAP-DLRIE, DOTAP, DOPE, DMEAP,DODMP, DOPC, DDAB, DOSPA, EDLPC, EDMPC, DPH, TMADPH, CTAB, lysyl-PE,DC-Cho, -alanyl cholesterol; DCGS, DPPES, DCPE, DMAP, DMPE, DOGS, DOHME,DPEPC, Pluronic, Tween, BRIJ, plasmalogen, phosphatidylethanolamine,phosphatidylcholine, glycerol-3-ethylphosphatidylcholine, dimethylammonium propane, trimethyl ammonium propane, diethylammonium propane,triethylammonium propane, dimethyldioctadecylammonium bromide, asphingolipid, sphingomyelin, a lysolipid, a glycolipid, a sulfatide, aglycosphingolipid, cholesterol, cholesterol ester, cholesterol salt,oil, N-succinyldioleoylphosphatidylethanolamine,1,2-dioleoyl-sn-glycerol, 1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine,palmitoylhomocystiene,N,N′-Bis(dodecyaminocarbonylmethylene)-N,N′-bis((-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)ethylenediaminetetraiodide;N,N″-Bis(hexadecylaminocarbonylmethylene)-N,N′,N″-tris((-N,N,N-trimethylammonium-ethylaminocarbonylmethylenediethylenetriaminehexaiodide;N,N′-Bis(dodecylaminocarbonylmethylene)-N,N″-bis((-N,N,N-trimethylammoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diaminetetraiodide;1,7,7-tetra-((—N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptaneheptaiodide; orN,N,N′,N′-tetra((-N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N′-(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetriaminetetraiodide.

Actuators include all devices capable of compressing the deformablefluid reservoir to an extent that the macrostructures will move throughthe pores to the cells. Thus, suitable actuators will include mechanicalactuators (e.g., piston, screw drive, solenoid, etc.), hydraulicactuators (e.g., using aqueous fluid or oil), or pneumatic actuators(e.g., femtojet), even manual actuators. Therefore, it should also beappreciated that the actuator may be replaced by a pressurization devicethat directly pressurizes the contents of the deformable fluidreservoir, and suitable devices may include fluid pumps and pneumaticpumps. In still other aspects of the inventive subject matter, thecompression is directly performed on the cells and may this includeplanar pressure elements, rollers, etc., or pressurized fluid dropletsenclosing macrostructures may be accelerated onto the cells (e.g., viafluid jet devices, or inkjet print heads, etc.), typically at pressuresof less than 1000 hPa.

In still further preferred aspects, the actuator operation is performedvia a controller that is configured to move the actuator such thatmovement of the actuator effects deformation of the deformable fluidreservoir, and with that pressurizes the fluid. The inventors havediscovered, as is shown in more detail below, that cell transfection isparticularly effective in a relatively small range of conditions. Inespecially preferred aspects, the controller is configured to applypressure via the actuator for a period of time ranging from about 10 msto about 30 s, or from about 20 ms to about 15 s, or from about 20 ms toabout 300 ms. Thus, the controller will typically operate the plungersuch that the deformable fluid reservoir is pressurized for at leastabout 10 ms, at least 25 ms, at least 50 ms, at least 100 ms, or atleast 500 ms, or at least 1 s, or at least 5 s, but in most cases lessthan 10 s, or less than 20 s, or less than 30 s, or less than 60 s. Insome instances, pressurization may also last about 1 min, or up to about1.5 min, or up to about 2 min, or up to about 2.5 min, or up to about 5min. Most typically, however, the controller will be configured toeffect pressurization for a period of time ranging from about 10 ms upto about 500 ms.

The controller is also preferably configured such as to effectpressurization of the fluid in the deformable fluid to a pressure of atleast 10 hPa, at least 20 hPa, at least 50 hPa, at least 100 hPa, atleast 200 hPa, at least 400 hPa, or at least 800 hPa. Therefore,suitable pressure ranges effected by the controller will be between 10hPa and 1000 hPa, or between 20 hPa and 800 hPa, or between 40 hPa and400 hPa, or between 50 hPa and 500 hPa. Additionally, it should beappreciated that the slope of pressure increase may vary considerably,and in most cases maximum pressure levels will be attained within lessthan 10 s, or less than 5 s, or less than 1 s, or less than 500 ns, orless than 250 ms. Of course, it should be recognized that the controllermay also be programmable to a particular profile having a predeterminedmaximum pressure, predetermined pressure duration, and/or predeterminedtime to maximum pressure. Where feedback mechanisms are contemplated, itis typically preferred that the controller receives information of atleast one pressure sensor in the device, typically located in thedeformable fluid reservoir.

FIG. 3 schematically and exemplarily illustrates two different modes ofactuation as discussed above. On the left panel, the actuator 340A is apneumatic actuator that provides pressurized air into the deformablefluid reservoir that has a flexible membrane 322A that forms part of thefluid reservoir above (container not shown). In such embodiment, thedeformable portion is part of an inflatable chamber, the top of whichforms the bottom of the fluid reservoir. Similarly, on the right panel,the actuator 340B is a solenoid actuator that directly presses againstthe deformable membrane 322B, the top of which forms the bottom of thefluid reservoir. As will be readily appreciated, the type of actuatorand controller settings all fine-tuned pressure increases as can betaken from FIG. 4. Here, the graph depicts deflection kinetics of thedeformable portion of the fluid chamber as a function of operation andtype of actuator.

Experimental Data

The following experiments provide exemplary guidance on certain aspectsof the device and methods of use, but should not be construed to belimiting in any manner. Unless specified otherwise, all transfectionexperiments were performed with a device according to FIG. 1A using thefollowing typical experimental set-up:

Recipient cells are cultured or immobilized on top a 10 μm-thickpolycarbonate porous membrane. For mitochondria delivery, membranes with3 μm pore diameter and a density of 2×10⁶ pores/cm² are used. For RNAand DNA lipoplex delivery, membranes with 1 μm pore diameter and 1.6×10⁶pores/cm² are used. Cargo suspension is loaded in a fluid reservoir madeby stacking two layers of polydimethylsiloxane (PDMS). Bottom layer hasa thickness of 0.5 mm and top layer has a thickness of 1 mm. Reservoirvolume is 100 μL. The porous membrane is clamped and sealed onto thePDMS reservoir which connects the recipient cells with cargo suspensionvia its pores. A motorized actuator (solenoid actuator or stepper motor)is affixed to the bottom of the PDMS fluid reservoir. For delivery, theactuator is activated to deform the fluid reservoir and subsequentlypump the cargo suspension into the recipient cells. For mitochondriadelivery, a flow rate of 100 μL/0.02 sec is applied whereas for lipoplexdelivery, a flow rate of 100 μL/10 sec is applied. After delivery,recipient cells can be cultured further on the porous membrane orre-plated onto other substrates for expansion or analyses. In theexperiment demonstrating reprogramming human fibroblast into inducedpluripotent cells, reagents and reprogramming protocol are described byP. Mandal and D. Rossi in Nature Protocols 8, 568-582 (2013). In theexperiment demonstrating CRISPR genome editing, reagents and guidesequence construction are described by F. Ran et al. in Nature Protocols8, 2281-2308 (2013).

More specifically, FIG. 5 exemplarily illustrates suitable pumppressures to achieve transfection of mammalian cells with mitochondria,while FIG. 6 provides a comparison for transfection efficiency using thedevices presented herein vis-à-vis conventional transfection methods.FIG. 5 presents graph that shows the number of successful mitochondriatransfected colonies generated at various pressures. When the assemblyis not clamped or locked down, no successful colonies were generated.Interestingly, at ambient pressure where the device is only under clamppressure transfection occurs. Thus, it should be appreciated that thedevices contemplated herein are able to achieve successful transfectionat pressures that are at about ambient pressure, at least 50 hPa aboveambient pressure, at least 100 hPa above ambient pressure, 200 hPa aboveambient pressure, or even at least 400 hPa above ambient pressure. FIG.6 illustrates a comparison of the transfection device contemplatedherein with various other techniques. As can be readily seen from thegraph, the device according to the inventive subject matter is capableof successfully transfecting cells with mitochondria as compared toother known techniques that failed to deliver mitochondria at anysignificant fraction. The data in FIG. 6 were generated using a PASTe(Pressure Assisted Cell Surgery Tool) device that has a porous substratewith 3 μm pores.

The colony forming frequency for mitochondria delivery (Fc) iscalculated by dividing the number of transfected cells (Nt) thatsuccessfully grow colonies by number of initial cells (Ni); thusFc=Nt/Ni. Interestingly, just after delivery, both the PASTe device andco-incubation delivered mitochondria with an efficiency of about 80%.However, only the PASTe device generated transfected cells capable ofgrowing colonies that remained alive and inherited the transfectedmitochondria.

FIG. 7 shows exemplary results for various porous materials that can beused with the devices according to the inventive subject matter. As canbe readily seen from the graph, a broad spectrum of porous substratesincluding at least PC, PET, deformable membranes, or even rigidsubstrates such as silicon can be successfully employed. Thesesubstrates are readily available from manufactures including CORNING®,BECTON DICKENSON®, and WHATMAN®. Furthermore, the graph also indicatesthat transfection with mitochondria is successful with a range poresizes from 0.4 μm through at least 8 μm. Most notably, the data alsoindicate that mitochondria delivery via contemplated transfectiondevices can have a success frequency that is at least 1×10⁻⁵, morepreferably at least 8×10⁻⁵, even more preferably at least 1×10⁻⁴, or yetmore preferably at least 4×10⁻⁴. Liposome delivery has successfullyachieved efficiencies of at least 15%, including at least 70%.

FIG. 8 presents data using pore density for the substrates from FIG. 7.As can be readily taken from the data, acceptable pore densities(pores/cm²) be a least 1×10⁴ pores/cm², at least 1×10⁵ pores/cm², atleast 4×10⁶ pores/cm², at least 1×10⁷ pores/cm², or even at least 1×10⁸pores/cm². When the data from FIGS. 7 and 8 are combined an indicationcan be obtained which substrate will generate the most viable coloniesas shown in FIG. 9 in which colony frequency has been normalized by poredensity. At least with respect to mitochondria delivery and the set ofsubstrates tested, an 8 μm pore size having relatively low pore densityyields the most viable colonies at least for the experiments run.

FIG. 10 exemplarily shows results for transfection using DNA and RNAlipoplexes in conjunction with the devices contemplated herein as wellas with traditional top transfection in which DNA and RNA lipoplexeswere applied on top of MDCK cells. The delivered lipoplex contains greenfluorescent protein (GFP) encoding DNA or RNA. As a result, successfullytransfected cells are indicated by their GFP expression. As used herein,the term “lipoplex” refers to a composition that includes a nucleic acid(RNA or DNA) in association with a lipid, and most typically a cationiclipid that is in most cases organized as a micelle or liposome. Cellmortality after transfection is measured by propidium iodine (PI)staining. Percentage of PI positive cells represents the dead cellpopulation. Cells were plated on day 0 and transfection was carried outon day 1, 2 and 3 respectively. PASTe yielded higher transfection ratethan traditional applying lipoplex to top of the cells in allexperiments, especially for higher cell densities (day 3). The PASTeapproach (using a device as shown in FIG. 1) used a 1 μm pore substrateand a 3 μm pore substrate, and it is readily apparent that PASTe devicesoutperformed the traditional techniques.

The inventors further conducted experiments to investigate the influenceof actuator speed on transfection efficiency. As can be seen from theTable below, transfection efficiency with mitochondria is a function ofactuator speed.

Cell density pump speed after delivery Colony # with Pump method (mm/s)(per uL) delivered mtDNA Solenoid 100 39 252 Pneumatic pump 10 53.6 135Stepper motor 2 86.3 29 Stepper motor 0.1 76.4 3

In contrast, the following table below indicates the results for DNALipoplex PASTe as a function of pumping speeds. Notably, however, wherethe transfection used nucleic acids, the transfection efficiency was notmaterially affected by actuator speed.

pump speed GFP expression Median GFP Pump method (mm/s) (%) intensitySolenoid 100 29.3 12,256 Femtojet 10 41.4 11,421 Stepper 1 39.6 11,944Top down — 32.6 3,346 transfection

To further investigate the role of mitochondria status in celltransfection, the inventors compared fresh isolated mitochondria,previously frozen mitochondria, and mtDNA using the devices according tothe inventive subject matter. Here, FIGS. 11A and 11B illustrate thatthe state of the isolated mitochondria is not critical for transfectionresults, nor that transfection efficiency is critically affected byvarious mitochondria inhibitors (CCCP: drug to dissipate mitochondrialmembrane potential; Oligomycin: inhibits mitochondrial respiration; R6G:inhibits mitochondrial respiration).

Further experiments, as exemplarily depicted in FIGS. 12A and 12B,suggest that the uptake of mitochondria is an active and energydependent process. Here, transfection efficiency is significantly lowerat lower temperatures. Amiloride is a macropinocytosis inhibitor,Genistein and Cyclodextrin are caveolae mediated endocytosis inhibitors,and Chlorpromazine is a clathrin mediated endocytosis inhibitor. As canbe seen from these experiments, amiloride significantly inhibited colonyformation and thus suggests an active update mechanism (e.g., viaendocytosis, and especially macropinocytosis).

FIGS. 13A and 13B illustrate exemplary results from transfection ofhuman skin fibroblasts comparing various transfection agents and methodsfor RNA. Here, GFP mRNA was complexed with various transfection reagents(Lipofectamine 2000, Lipofectamine RNAiMAX, and Stemfect) according tothe manufacturer's protocols prior to delivery into the cells via topdown transfection or PASTe. As is readily apparent not all transfectionagents had the same effect, and the best agent tested was LF2k(Lipofectamine 2000) for PASTe devices, while stemfect was best for toptransfection in terms of transfection efficiency as well as expressionlevels. Additionally, it should be appreciated that transfectionefficiency as well as expression levels using PASTe were superior toconventional down transfection. Thus, cells can be effectivelytransformed using RNA to express recombinant protein at relatively highlevels and transfection efficiency.

In yet another set of experiments, the inventors employed contemplateddevices and methods to increase efficiency and reproducibility forreprogramming human fibroblasts into induced human pluripotent stem(iPS) cell. Here, mRNA cocktails containing reprogramming factors OCT4,KLF4, C-MYC, SOX2 and LIN28A were complexed with different transfectionreagents according to manufacturer's protocol and delivered into humanskin fibroblasts daily over a course of 2 weeks using top downtransfection (P. K. Mandel and D. J. Rossi. Reprogramming humanfibroblasts to pluripotency using modified mRNA. Nature Protocols 8, p.568-582, 2013) and PASTe transfection. At the end of two weeks,successfully reprogrammed iPS colonies expressing pluripotent markerswere counted and compared between different experimental conditions. Ascan be taken from the results in the table below, contemplated PASTedevices and methods were once more superior to traditional transfectionusing transfection agents without the device. Thus, treatment of cellswith multiple distinct RNA species in association with transfectionagents provided remarkably high transfection efficiency and expressionlevels (as can be taken from the FACS plots).

Well #4 Well #1 Well #2 Well #3 LF2k + PASTe LF2k RNAiMAX LF2k (ramp)miRNA iPS colony # 29 21 23 73 at D14 Well #4 Well #1 Well #2 Well #3Stemfect + Top LF2k RNAiMAX Stemfect miRNA iPS colony # 0 3 0 0 at D14

As can also be seen from the data, the mean reprogramming efficiency forPASTe is between 0.1-0.4%, while the mean reprogramming efficiency forTop transfection is about 0.015% Likewise, the success rate for PASTe is100%, while the success rate for Top down transfection is about 25%.Such benefits are unexpected as heretofore transfection agents alonewere deemed critical to the success of the transfection and not thespecific manner of delivery. FIG. 14 provides a comparison and contrastof a “Top” approach relative to the inventive PASTe approach for RNAtransfection into BJ fibroblasts. Note that the PASTe approach not onlyprovides for a significant number of transfection events relative to theTop approach, but also generates transfected cells that are highlydifferentiable from non-transfected cells (control group as shown inleft panel). Thus, transfected cells can be selected based on FL1-H at ahigher efficiency relative to transfected cells generated by the Topapproach.

FIG. 15 provides an example where systems and methods according to theinventive subject matter were employed with H9 human embryonic stemcells (hESC). Here, the relative efficacy of a PASTe approach comparedto a Top approach is shown for RNA transfection. Note that, as in FIG.14, transfected hESC are more effectively differentiated fromnon-transfected cell based on FL1-H. Thus, systems and methods accordingto the inventive subject matter allow for a significantly more efficientmechanism for genomic manipulation and subsequent identification oftransfected cells (e.g., reprogrammed cells, stem cells, etc.) thanother known approaches.

As noted above with respect to FIG. 14, differentiated cells (e.g., skinfibroblasts) can be reprogrammed to induced pluripotent (iPS) cells bydaily transfection of five transcription factors (OCT4, KLF4, C-MYC,SOX2, LIN28A) for a duration of approximately 14 days using heretoforeknown protocols. Unfortunately, such time frames can be unduly long. Inorder to determine the viability of PASTe with respect to time framesfor genomic manipulation, the inventors followed the protocol describedby P. Mandel and D. Rossi (Nature Protocol 8, 2013; see FIG. 1) andtransfected BJ fibroblast cells with mRNA cocktails encoding KOMSLfactors for 14 days. For comparison, mRNAs lipoplexes were delivered toBJ cells grown with irradiated feeder cells via the PASTe deliveryapproach as well as a conventional top transfection approach forcomparison. As can be seen in FIG. 16, stem-cell-like colonies in PASTetransfected cells are produced as early as day 8. It should also benoted that by day 5, using the PASTe approach, stem-cell-like coloniesalready began to appear. The resulting PASTe colony sizes were largerand denser than those by Top transfection, and the morphology of thecells appeared to more closely resemble fully reprogrammed iPS cellcolonies.

Interestingly, genome manipulation through PASTe RNA lipoplextransfection does not appear to depend on a pump speed (e.g., impulsegenerated), while PASTe mitochondrial transfer did appear to depend onpump speed as is shown in the tables below. The observed lipoplextransfection efficiency (e.g., GFP expression percentage) using thePASTe approach was relatively independent of pump speed. However, thePASTe mitochondria transfer and colony forming efficiency appeared todepend on pump speed. Thus, PASTe devices that are configured for genomemanipulation via lipoplex-based transfection can adapt a pump method tofit a target circumstances (e.g., time, cost, latency, etc.).

PASTe Transfection (RNA lipplex) of hESC at different pump speed PumpPump Speed GFP Median Method (mm/s) Expression Mean GFP GFP Solenoid 10029.3 18,015 12,256 Femtojet 10 41.4 17,297 11,421 Stepper 1 39.6 17,18211,944 Top Transfection -/- -/- 32.6 7,474 3,346 PASTe Transfection(Mitochondria) of 143btk rho(0) cells at different pump speed Pump PumpSpeed Cell density/μL Method (mm/s) (after delivery) Uptake (%) Colony #Solenoid 100 39 12.4 252 Femtojet 10 53.6 61.3 135 Stepper 2 86.3 65.629 Stepper 0.1 76.4 61.6 3

Contemplated methods and devices can also be employed for transfectionwith complex cargo. For example, cells can be transfected with DNA andRNA in an approach to correct or change genomic information (e.g., viahomology directed repair, genome editing via Crispr/Cas, etc.). Asbefore, the different types of nucleic acids are preferably associatedwith respective transfection reagents prior to contacting the cells withthe cargo. FIG. 17 schematically shows a PASTe-CRISPR genome editingapproach in which the DNA portion comprises a plasmid that encodes theguide RNA, Cas9, and OFP, while the RNA portion includes single strandednucleic acid HDR template. The components may be combined or separatelybe associated with the transfection agent (e.g., lipofectamine 2000) andwere delivered into HEK293 cells using PASTe or Top down transfection.As before, PASTe transfection showed a significantly increasedtransfection efficiency as compared to Top down transfection and highergene editing efficiency by homology directed repair. FIG. 18 providesexemplary results for transfection efficiency in HEK 293 cells, and FIG.19 exemplarily demonstrates results for the gene editing efficiency byhomology-directed repair (HDR).

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of changing genetic content of a cell invitro, comprising: providing a plurality of cells in vitro and exposingthe cells to a transfection medium comprising a transfection reagent inassociation with a plurality of distinct nucleic acids, wherein theplurality of cells, during the step of exposing, are located on asynthetic porous membrane; wherein the step of exposing the cellscomprises applying fluid pressure to the transfection medium frombeneath the synthetic porous membrane to the cells to thereby force thetransfection medium against the cells through the synthetic porousmembrane for a time and at a pressure effective to introduce theplurality of distinct nucleic acids into the cells by endocytosis; andcultivating the cells after the step of exposing for a time sufficientto integrate or express the plurality of distinct nucleic acids.
 2. Themethod of claim 1 wherein the step of exposing the cells isnon-ballistic and wherein the pressure to the transfection medium is atleast 100 hPa.
 3. The method of claim 1 wherein the pressure to thetransfection medium is maintained for a period of between 10 ms and 30s.
 4. The method of claim 1 wherein the plurality of cells are retainedin a fixed position during the step of exposing.
 5. The method of claim1 wherein the plurality of cells are retained using a microfluidicchannel or a cell adhesive layer.
 6. The method of claim 1 wherein thefluid pressure is applied uni-directionally from beneath the syntheticporous membrane to the cells via direct or indirect actuation.
 7. Themethod of claim 6 wherein the porous membrane has an average poredensity of between 1×10⁶ pores/cm² and 1×10⁸ pores/cm².
 8. The method ofclaim 7 wherein the porous membrane has an average pore size of between0.5 μm and 10 μm.
 9. The method of claim 1 wherein the steps of exposingand cultivating are performed in the same container containing theplurality of cells.
 10. The method of claim 1 wherein the change ingenetic content reprograms a differentiation stage of a cell or editsgenomic information of a cell.
 11. The method of claim 1 wherein thecells are fully differentiated cells, and wherein the change in geneticcontent reprograms the cells to be induced pluripotent cells.
 12. Themethod of claim 1 wherein the change in genetic content reprograms thecells; and wherein after reprogramming the cells are genomically editedcells.
 13. The method of claim 1 wherein the transfection mediumcomprises cationic liposomes.
 14. The method of claim 1 wherein theplurality of nucleic acids comprise a guide RNA or a regulatory RNA. 15.The method of claim 1 wherein the plurality of nucleic acids comprise aplurality of distinct RNAs that encode respective different proteins.16. The method of claim 15 wherein the RNAs encode genes suitable forreprogramming a differentiated cell into a induced pluripotent stemcell.
 17. The method of claim 16 wherein the induced pluripotent stemcell is a human cell.
 18. The method of claim 1 wherein the plurality ofnucleic acids comprise at least one DNA and at least one RNA.
 19. Themethod of claim 18 wherein the at least one DNA and the at least one RNAare suitable for genome editing.
 20. The method of claim 1 wherein thecells, after integrating or expressing the plurality of distinct nucleicacids, are cultivated for a time sufficient to generate a clonaldaughter cell.
 21. The method of claim 2, wherein the pressure to thetransfection medium ranges from 100 hPa to 400 hPa.
 22. The method ofclaim 1, wherein the change in genetic content reprograms the cells; andwherein the mean reprogramming efficiency of the reprogrammed cells isbetween 0.1-0.4%.
 23. The method of claim 1, wherein colonies ofcultivated cells are detected by at least five days after exposing thecells.